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. 2019 Dec;26(1):965-974.
doi: 10.1080/10717544.2019.1667453.

Delivery of antisense oligonucleotide using polyethylenimine-based lipid nanoparticle modified with cell penetrating peptide

Shuang Yang  1 , Dandan Wang  2 , Yaojun Sun  1 , Bin Zheng  3
Affiliations

Delivery of antisense oligonucleotide using polyethylenimine-based lipid nanoparticle modified with cell penetrating peptide

Shuang Yang et al. Drug Deliv. 2019 Dec.

Abstract

Efficient and stable delivery system of antisense oligonucleotide (ASO) is important and urgently needed. Here, an ASO delivery system, Lp-PPRP, which contains a cationic polymer based on PEI (branched, 25 kDa), named PEI-PC and a palmitic acid modified R8 (R8-PA) was prepared to deliver a kind of ASO, LOR-2501. The characteristics of the nanoparticles and the cellular uptake of LOR-2501 in HeLa cells and A549 cells were studied. Lp-PPRP showed suitable particle size and zeta potential to combine with LOR-2501; the particle size and zeta potential of Lp-PPRP/LOR were 276.87 ± 5.63 nm and 18.03 ± 0.25 mV. In vitro experiments suggested that Lp-PPRP had lower cytotoxic and higher transfection efficiency for delivering LOR-2501 compared with PEI. The addition of PEI-PC and R8-PA contributed to enhance the transfection efficiency of the nanoparticles. In HeLa cells and A549 cells, Lp-PPRP could transport LOR-2501 and down-regulate the level of R1 protein efficiently, and the R1 down regulations were 64.56% and 66.34%, respectively. Results suggested potential utility of Lp-PPRP in the development of ASO in tumor therapy.

Keywords: Polyethylenimine; antisense oligonucleotide; cancer; cell penetrating peptide; lipid nanoparticles.

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Figures

Figure 1.
Figure 1.
The structural formulae of PEI-PC, R8-PA, and schematic diagram of the preparation process of Lp-PPRP/LOR. (A) The structural formulae of PEI-PC. (B) The structural formulae of R8-PA. (C) The schematic diagram of the preparation process of Lp-PPRP/LOR.
Figure 2.
Figure 2.
Mean sizes and zeta potentials of Lp-PP/LOR and Lp-PPRP/LOR. (A) Mean sizes of Lp-PP/LOR at varying N/P ratios from 2:1 to 12:1. (B) Zeta potentials of Lp-PP/LOR at varying N/P ratios from 2:1 to 12:1. (C) Mean sizes of Lp-PPRP/LOR at varying R8-PA/total lipid ratios from 5% to 30%. (D) Zeta potentials of Lp-PPRP/LOR at varying R8-PA/total lipid ratios from 5% to 30%. ‘N’ represented the molar weight of nitrogen in PP and ‘P’ represented the molar weight of phosphorus in LOR.
Figure 3.
Figure 3.
Agarose gel electrophoresis retardation assay of Lp-PP/LOR, Lp-PPRP/LOR and the characterization of Lp-PPRP/LOR. (A) Agarose gel electrophoresis retardation assay of Lp-PP/LOR at varying N/P ratios. (B) Agarose gel electrophoresis retardation assay of Lp-PPRP/LOR. (C) FE-SEM image of Lp-PPRP/LOR at R8-PA/total lipid ratio = 20%. (D) Diameter distribution of Lp-PPRP/LOR at R8-PA/total lipid ratio = 20%. (E) Zeta potential distribution of Lp-PPRP/LOR at R8-PA/total lipid ratio = 20%.
Figure 4.
Figure 4.
Cytotoxicity tests of PEI, PP, LP-PP and LP-PPRP. (A) Cytotoxicity tests of PEI, PP, LP-PP, and LP-PPRP on HeLa cells. (B) Cytotoxicity tests of PEI, PP, LP-PP, and LP-PPRP on A549 cells. Each bar is the mean of six experiments normalized to mean ± SD. *p < .05 vs. control and **p < .01 vs. control.
Figure 5.
Figure 5.
Cellular uptake of 5′-Cy3-labled LOR-2501 delivered by Lp-PEI, Lp-PP, and Lp-PPRP. (A) Cellular fluorescence uptake of Lp-PEI/LOR, Lp-PP/LOR, and Lp-PPRP/LOR by flow cytometry in HeLa cells and A549 cells. (B) The mean fluorescence values of HeLa cells treated with Lp-PEI/LOR, Lp-PP/LOR, and Lp-PPRP/LOR. (C) The mean fluorescence values of A549 cells treated with Lp-PEI/LOR, Lp-PP/LOR, and Lp-PPRP/LOR. LOR-2501 was labeled with 5′-Cy3. **p < .01 vs. Lp-PEI/LOR and ##p < .01 vs. Lp-PP/LOR.
Figure 6.
Figure 6.
Down regulation of R1 protein expression in HeLa cells and A549 cells treated with Lp-PEI/LOR, Lp-PP/LOR, and Lp-PPRP/LOR. (A, C) HeLa cells. (B, D) A549 cells. *p < .05 vs. Lp-PEI/LOR, **p < .01 vs. Lp-PEI/LOR, #p < .05 vs. Lp-PP/LOR, and ##p < .01 vs. Lp-PP/LOR.
Figure 7.
Figure 7.
Intracellular localization of 5′-Cy3-labled LOR-2501 delivered by Lp-PEI, Lp-PP, and Lp-PPRP in HeLa cells shown by confocal microscopy. 5′-Cy3-labled LOR-2501 is shown in red, DAPI nuclear stain is shown in blue. Scale bar = 20 μm.
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